Crude biological derivatives competent for nucleic acid detection

ABSTRACT

The invention relates to methods for the detection of a specific sequence of RNA in a cell or tissue sample. The invention also relates to methods to enzymatically manipulate the RNA in a crude cell lysate in a number of applications.

The present application claims priority to pending U.S. application Ser.No. 15/466,824 filed Mar. 22, 2017, which is a continuation of U.S.application Ser. No. 13/463,624 filed May 3, 2012 (now U.S. Pat. No.9,611,497), which application is a continuation application of U.S.application Ser. No. 10/352,806 filed Jan. 28, 2003 (now abandoned),which application claims the benefit of U.S. Provisional Application No.60/352,402 filed Jan. 28, 2002. The entire texts of the above referencedapplications are incorporated herein by reference and withoutdisclaimer.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of RNA analysis,more specifically it teaches a more direct method for the detection of aspecific sequence of RNA in a biological unit, for example a virus, cellor tissue sample. More generally, the invention may be used toenzymatically manipulate and protect the RNA in a crude cell lysate fora number of applications.

2. Description of Related Art

Reverse transcription followed by the polymerase chain reaction (RT-PCR)is one of the main methods used for measuring mRNA levels from a smallnumber of cells. As well, reverse transcription is the first step inseveral strategies towards amplifying a small quantity of total or poly(A) RNA (U.S. Pat. No. 5,554,516; U.S. Pat. No. 5,891,636; Phillips,1996). The amplified RNA can be used to probe arrays for monitoring theexpression of multiple genes (Lockhart, 1996; U.S. Pat. No. 6,316,608).Prior to performing any of these methods, the substrate RNA is isolatedfrom a biological sample, in most cases. Current procedures for RNAisolation involve numerous steps and are not very amenable to highthroughout analysis.

In general, the techniques used for RNA isolation involvephenol-chloroform extraction (Mesink, 1998) or guanidinium lysisfollowed by adsorbing the RNA to a glass fiber filter (Su, 1997). Bystreamlining the RNA isolation step, the analysis of a large number ofsamples involving reverse transcription or some other enzymaticmanipulation becomes much faster, simpler, and less expensive. Klebe etal. (1996) developed a strategy of creating a crude cell lysate byfreeze-thawing cells at a concentration of 10 cells/μl in the presenceof placental RNase inhibitor. The crude lysate, containing no more than250 cells, was then used for reverse transcription to produce cDNA. Thistechnology serves as the basis for the “cDNA Direct from Cells” kit soldby PCG (Cat. #62-613100). As pointed out by Klebe (1996), this method islimited in that the RNase inhibitor is only specific for RNase A. Thereare many other types of RNases in a cell that may contribute to RNAdegradation and would not be inhibited by a single specific RNaseinhibitor. Another problem is that some types of cells have a muchhigher concentration of RNase activity thereby making it more difficultto maintain the intactness of the RNA in a crude lysate (O'Leary, 1999).A similar protocol was used by Yan. (2002) to detect an mRNA from onecell by RT-PCR. However, it differed in that it also included a DNasetreatment to remove genomic DNA and only 1 to 3 cells were used in thereactions.

Busche (2000) used a procedure similar to Klebe (1996) to reversetranscribe RNA from a few cells. Ten myocyte section profiles fromvarious samples were selected by laser-assisted picking, transferredinto 10 μl of first strand buffer containing 4% ribonuclease inhibitor,cooled on ice for 5 minutes and snap frozen. The samples were incubated70° C. for 10 minutes and cooled on ice for 5 minutes. Reversetranscription was performed in a total of 17.5 μl using 5 μl of thesample and an MMLV-RT, and incubated 20° C. for 10 minutes followed by43° C. for 60 minutes. The cDNA was subsequently used for PCR.

Brady (1993) generated cDNA from a few cells for creating cDNA banksusing lysed cells. One to 40 cells in less than 0.5 μl volume are addedto 4 μl of first-strand buffer and stored on ice for less than one hourbefore reverse transcription. The first strand buffer contains 0.5%Nonidet P-40 (NP-40) to lyse the cellular membrane and an RNaseinhibitor to protect the RNA from degradation. The NP-40 does not lysethe nuclear membrane and therefore, the nucleus can be pelted bycentrifugation (e.g., centrifugation at 12,000×g, 4° C., for 50seconds), to deplete the cell lysate of genomic DNA if desired. Thecytoplasmic RNA is used for reverse transcription. The cell lysate inthe first strand buffer is incubated at 65° C. for 1 minute to unfoldthe mRNA. The reaction is cooled to room temperature for 3 minutes toanneal the oligo (dT) primer. One μl of a 1:1 mix of MMLV-RT and AMV-RTis added to the reaction and incubated 15 minutes at 37° C. The reactionwas stopped by heating to 65° C. for 10 minutes. This procedure does notinvolve any protease treatment, any DNase treatment and is onlyrecommended for no more than 40 cells.

A kit called ExpressDirect™ (Pierce Chemical Company, Cat. #20146),isolates poly(A) RNA directly from a cell lysate. The wells of a 96-wellplate have oligo dT immobilized to them. Cells are lysed in the wellsand the poly(A) RNA hybridizes to the oligo dT. After hybridization, thecell lysates are removed and the wells washed to remove cell debris. Thepoly(A) RNA may then be eluted from the well and then reversetranscribed. Alternatively, the poly(A) RNA could be reverse transcribeddirectly in the 96-well plate. The immobilized oligo dT serves as theprimer.

Protocols exist for the detection of bacterial DNA sequences from tissueculture in order to assay for Mycoplasma contamination (U.S. Pat. No.5,693,467; and Tang, 2000). This procedure involves incubating the cellsfrom tissue culture with proteinase K. However, there is no mention ofusing this procedure to synthesize cDNA. In the Mycoplasma Detection kitfrom the American Type Culture Collection (Cat. #90-1001K) cells to betested for Mycoplasma from tissue culture can be subjected directly toPCR if the Mycoplasma contamination is suspected to be severe. However,to achieve maximum sensitivity the cells are incubated in a lysis buffer(lx PCR buffer, 0.5% NP-40, 0.5% Tween 20) with proteinase K (18 μg/ml)at 60° C. for one hour. The lysate is then incubated at 95° C. for 10minutes to inactivate the proteinase K. The manual states that the DNAextract may be used directly as the template for PCR without furtherpurification. However, it cautions that the completion of the secondaryDNA extraction procedure facilitates removal of all possible PCRinhibitors. The secondary extraction protocol involves adding 500 μlwater, mixing well, adding 600 μl isopropanol and 1 μl glycogen (20mg/ml), mixing well, incubating at −20° C. for at least 30 minutes,centrifuging to pellet the DNA and then removing the supernatant. TheDNA pellet is washed with 75% ethanol, centrifuged again and thesupernatant removed. No mention is made in that this procedure can beused to prepare RNA for reverse transcription.

Fink (2000a; 2000b) used a proteinase K treatment to increase theefficiency of RT-PCR from cells isolated by laser-assisted cell picking.Between 15 and 20 frozen or fixed cells were selected by laser-assistedcell picking, harvested by a syringe needle, added to 10 μl offirst-strand-buffer and frozen in liquid nitrogen. After thawing thecells, proteinase K was added to the sample to 100 μg/ml, the sample wasincubated at 53° C. for 30 minutes and then heated at 99° C. for 7minutes to denature the proteinase K and RNA. Reverse transcription wasperformed directly on the sample using murine maloney leukemiavirus-reverse transcriptase (MMLV-RT), at 20° C. for 10 minutes and 43°C. for 60 minutes. The cDNA from this reaction was used for PCR. In bothof these publications, the fixed cells were frozen before the proteinaseK treatment, the concentration of cells was no more than 2 cells/μl andno DNase treatment was used to remove genomic DNA.

Cells to cDNA™ (Ambion, Inc., #1712 & 1713; U.S. patent application Ser.Nos. 09/160,284 and 09/815,577, the entire disclosures of which areincorporated herein by reference) is a kit where there is no RNAisolation step. A crude cell lysate is prepared containing total RNA.Cells from tissue culture are washed once in PBS and then resuspended inCell Lysis Buffer. The cells are incubated at 75° C. for 5 minutes,having two important effects. First, the cell membranes are lysed,thereby releasing the RNA into the Cell Lysis Buffer. As well, theheating step inactivates the endogenous RNases, thus protecting the RNAfrom degradation. A key component in the Cell Lysis Buffer is a reducingagent such as dithiothreitol (DTT). It was discovered that RNases can beinactivated by heating them in the presence of reducing agents (U.S.patent application Ser. Nos. 09/160,284 and 09/815,577). Following celllysis, the crude cell lysate is incubated with DNase I to degrade thegenomic DNA. After the DNase I is inactivated by a heating step, thecell lysate is ready for reverse transcription and then PCR. TheCells-to-cDNA™ kit (Ambion, Inc. Cat. #1712 & 1713) is adapted for usewith samples having low cell concentrations. If higher cellconcentrations are used, then RNA quantification can cease to be linearand in some cases, the signal can be completely inhibited. It appearsthat the reverse transcriptase can be inhibited by the higher cellconcentrations. In general, the maximum optimal cell concentration theCells-to-cDNA™ kits is 100 to 200 cells per μl in the Cell Lysis Buffer.

A procedure that enables the direct use of a cell lysate at a highercell concentration would have many more applications and provide agreater dynamic range for quantification, thereby complimenting thetechnology in Cells-to-cDNA. Also, because of the issue of higher cellconcentrations, Cells-to-cDNA is most useful in the context of cellsfrom tissue culture. As well, methods that are more useful in the directuse of a tissue in a reverse transcription reaction would decrease thetime and the amount of handling required to prepare a sample for reversetranscription or other enzymatic applications.

SUMMARY OF THE INVENTION

The above-described deficiencies in the art are overcome by the presentinvention.

Broadly, the present invention relates to methods comprising: obtainingat least one biological unit containing RNA; obtaining at least onecatabolic enzyme; preparing an admixture of the biological unit and thecatabolic enzyme; and incubating the admixture under conditions wherethe catabolic enzyme is active.

The term “biological unit” is defined to mean any cell or virus thatcontains genetic material. In most aspects of the invention, the geneticmaterial of the biological unit will include RNA. In some embodiments,the biological unit is a prokaryotic or eukaryotic cell, for example abacterial, fungal, plant, protist, animal, invertebrate, vertebrate,mammalian, rodent, mouse, rat, hamster, primate, or human cell. Suchcells may be obtained from any source possible, as will be understood bythose of skill in the art. For example, a prokaryotic or eukaryotic cellculture. The biological unit may also be obtained from a sample from asubject or the environment. The subject may be an animal, including ahuman. The biological unit may also be from a tissue sample or bodyfluid, e.g., whole blood, plasma, serum, urine or cerebral spinal fluid.

The catabolic enzyme can be any catabolic enzyme known to those of skillin the art as of the filing of this specification or at anytimethereafter. In some preferred embodiments, the catabolic enzyme is aprotease, for example, proteinase K. In other embodiments, the catabolicenzyme degrades carbohydrates, for example, amylase or cellulase. Insome embodiments, the catabolic enzyme degrades lipids, such as lipase.In other embodiments the catabolic enzyme degrades DNA, such as, forexample, bovine pancreatic DNase I. Of course, the various embodimentsof the invention may comprise the use of one, two, three, four, five,six, seven, or more different catabolic enzymes, in order to achieve thedesired goals of a given method. In some embodiments, any enzymes thatdigest DNA, lipids, fats, carbohydrates, connective tissue or any othermolecules, proteins, or biological compounds that inhibit enzymaticreactions, specifically reverse transcription or PCR are contemplated.In certain embodiments, a catabolic enzyme may or may not be included inthe Cell Lysis Buffer and in some case may be introduced before, afteror simultaneously with the Cell Lysis Buffer. For example, it isentirely possible, in RT-PCR embodiments of the invention, to use bothproteinase K to destroy RNase in a cellular extract in combination withone or more other catabolic enzymes to degrade other portions of thecellular extract to the benefit of the reaction. Of course, in suchcases, it may be necessary to balance the concentrations and/or timingof the addition of the various catabolic enzymes, in order to prevent,for example, the degradation of a cellulase by a proteinase. However,such balancing will be well within the skill of one of skill in the art,in view of this specification. Proteases that may be used in the methodsof the invention include, but are not limited to, Serine proteases thatinclude but are not limited to Trypsin, Chymotrypsin, Elastase,Subtilisin, Streptogrisin, Thermitase, Aqualysin, and carboxypeptidaseA, D, C, or Y; cysteine proteases that include but are not limited toPapain and Clostripain; acid proteases that include but are not limitedto Pepsin, Chymosin, and Cathepsin; metalloproteases that include butare not limited to Pronase, Thermolysin, Collagenase, Dispase; andvarious aminopeptidases and Carboxypeptidase A, B, E/H, M, T, or U. Insome embodiments of the invention, these proteases could be used inplace of proteinase K. It is possible that a mixture of proteases couldbe used instead of a single protease to generate a cell lysatecompatible with reverse transcription and PCR.

In certain embodiments, a protease and a DNase enzyme may beadministered simultaneously or in the same reactions. This simultaneoustreatment using proteases and DNase enzymes is an unexpected and novelfinding, as described below. In some embodiments, 1, 2, 3, 4, 5, 6, 7,or more catabolic enzymes may be included in a protease/DNasecomposition or reaction. For example, multiple proteases or co-proteasesmay be included with lipases, collagenases, nucleases, and virtually anyother enzyme that may be used to remove inhibitors of a reaction, e.g.,polymerization reactions.

In some aspects of the invention, preparing an admixture of thebiological unit and the catabolic enzyme is further defined ascomprising preparing an extract of the biological unit and preparing anadmixture of the extract of the biological unit and the catabolicenzyme. Further, preparing an admixture of the extract of the biologicalunit and the catabolic enzyme may comprise: first preparing the extract;and then mixing the extract with the catabolic enzyme. Alternatively,preparing an admixture of the extract of the biological unit and thecatabolic enzyme could comprise: first mixing the biological unit andthe catabolic enzyme; and then preparing the extract from the biologicalunit in the presence of the catabolic enzyme.

In some preferred embodiments, the invention relates to methods forproducing cDNA from one or more biological units, possibly differenttypes of biological units. In some embodiments, any enzyme that canutilize a nucleic acid or in particular RNA as template or substrate iscontemplated. In certain embodiments, an admixture may be incubated witha nucleic acid polymerase. In some embodiments, the nucleic acidpolymerase is a ribonucleotide polymerase, e.g., bacterial or viral RNApolymerase. Preferred embodiments may further comprise incubating theadmixture with reverse transcriptase under conditions to allow reversetranscription. Typically, the methods will further comprise amplifyingthe products of the reverse transcription, and these methods may furthercomprise incubating said admixture with a deoxyribonuclease prior to thereverse transcription reaction. In some preferred embodiments thecatabolic enzyme is a protease that is capable of inactivatingribonucleases in the admixture. For example, the protease may beproteinase K.

These embodiments have certain benefits in the context of RT-PCR, withregard to the issues described above. In such methods, a freezing stepis not required. Further, the admixture may contain 1, 5, 10, 100, 200,300, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, or 15,000 ormore cells/μl, as well as any concentration of cells between any two ofthese concentrations. Note that these concentration are for typicaleukaryotic cells. Since prokaryotic cells are typically a thousand timessmaller these concentration may be adjusted accordingly. Typically, itis the reverse transcriptase enzyme that is inhibited by the higher cellconcentrations of cell lysate. In certain embodiments, the upper limitof cell concentration may be increased by either using other catabolicenzymes or other methods or compositions to destroy the inhibitors ofthe RT or by using RTs that are less inhibited by the lysates. Inaddition, DNase I treatment can be included although this is notnecessary in all embodiments, for example, if the PCR primers aredesigned properly and the gene structure is amenable. In view of theseimprovements, the methods of the invention are well-suited to theanalysis of a large number of differentially treated samples grown intissue culture. For example, the regulation of an mRNA may be followedas cells are treated with increasing concentrations of a particularchemical (Sumida, 1999). Alternatively, cells may be treated with apanel of different drugs to screen for candidates that have the desiredeffect on a particular mRNA or a time course may be followed (Su, 1997).

Some embodiments of the invention further comprise adding an RNaseinhibitor to the admixture, in addition to any proteinase that inhibitsor degrades RNase. For example, the RNase inhibitor is anon-proteinaceous RNase inhibitor, such as ADP or a vanadyl complex.Proteinaceous inhibitors could also be used such as placentalribonuclease inhibitor or antibodies that inactivate specificribonucleases.

In some embodiments, the final concentration of the catabolic enzymeadded is between about 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 2, 3, 4, 5,10, 15, 20, or 25 mg/ml, as well as any concentration between any two ofthese concentrations. In some embodiments, the final concentration ofthe catabolic enzyme added is between about 0.00001, 0.0001, 0.001,0.01, 0.1, 1, 2, 3, 4, 5, 10, 15, 20, or 25 mg/ml, as well as anyconcentration between any two of these concentrations in the admixture,more preferably, between 0.001 and 2 mg/ml, and even more preferably,between 0.025 and 1 mg/ml.

Typically, the catabolic enzyme is comprised in a buffer compositionprior to admixing.

In some preferred embodiments, the admixture is incubated at between 0°C. and 75° C. However, this temperature may vary during the course ofthe procedure. In certain embodiments, the admixture may be incubated atbetween 0° C. and 100° C. Further, it is entirely possible to raise thetemperature to a point where the catabolic enzyme is ultimatelyinactivated. For example, proteinase K tends to be inactivated at around75° C. The inventors frequently place proteinase K containing reactionsin a water bath at 75° C., knowing that when the reaction reaches thistemperature, the enzyme activity will be destroyed, but that the benefitof the enzyme in destroying RNase will be achieved by that point.

In preferred embodiments, the invention is related to methods forproducing cDNA from one or more biological units comprising: obtainingat least one biological unit; obtaining at least one catabolic enzyme;preparing an admixture of the biological unit and the catabolic enzyme;incubating the admixture at a temperature where the catabolic enzyme isactive; and incubating with reverse transcriptase under conditions toallow reverse transcription. The components of this reaction can be anyof the components described above.

Other embodiments of the invention relate to kits for producing cDNAfrom a biological unit, comprising, in a suitable container: a buffer;and a catabolic enzyme. In some such kits, the buffer and the catabolicenzyme are comprised in the same container. The kits may furthercomprise a reverse transcription buffer, a reverse transcriptase, anddNTP mix. The kits may additionally contain a deoxyribonuclease. In somepreferred embodiments, the catabolic enzyme is proteinase K. The kitsmay further comprise an RNase inhibitor.

In some preferred embodiments, the kits for producing cDNA from abiological unit comprises, in one or more suitable container(s): abiological unit lysis buffer; a deoxyribonuclease; an RNase inhibitor; areverse transcription buffer; reverse transcriptase; dNTPs; and anArmored RNA® control. “Armored RNA” is a an Ambion trademark forribonuclease resistant RNA particles produced according to the methodsdisclosed in U.S. Pat. Nos. 6,399,307; 6,214,982; 5,939,262; 5,919,625;and 5,677124, the entire contents of which are incorporated herein byreference. These kits may further comprise a protease inhibitor, such asphenylmethylsulfonyl fluoride (PMSF), and/or a thermostable DNApolymerase.

Kits suitable for the practice of the methods described herein are soldby Ambion under the trademark Cells-to-cDNA II™.

In other aspects of the invention, a Cell Lysis Buffer comprising acatabolic enzyme, 1 mM CaCl₂, 3 mM MgCl₂, 1 mM EDTA, 1% Triton X100, and10 mM Tris pH 7.5 is contemplated. In certain embodiments, the catabolicenzyme is Proteinase K. In some embodiments, Proteinase K may is presentat a concentration of about 0.2 mg/ml.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Illustrates an example of measuring the levels of GAPDH mRNA andthe enterovirus Armored RNA® by real-time RT-PCR in the ABI 7700 indifferent concentrations of HeLa cells. The cells were processed usingthe methods of the invention.

FIG. 2. Illustrates an example of measuring the levels of GAPDH mRNA byreal-time RT-PCR in the ABI 7700 in different concentrations of HeLacells that were unfixed or fixed with 1% formalin for 1 hour. The cellswere processed using the methods of the invention.

FIG. 3. Illustrates an example of measuring the levels of 18S rRNA andtPA mRNA in HeLa cells that were incubated with different concentrationsof PMA by one-step, real-time RT-PCR in the ABI 7700. The cells wereprocessed in 96-well plates using the methods of the invention.

FIG. 4. Illustrates an example of analyzing Gene Silencing by siRNAusing Cells-to-cDNA II™ Automated Protocol. HeLa cells were transfectedwith gene specific siRNAs against CDC-2, c-jun, survivin, and GAPDH or anegative control (NC1). After 48 hours, cells were processed accordingto the Cells-to-cDNA II™ automated protocol, and analyzed by real-timeone-step RT-PCR for the indicated genes and normalized to 18S rRNA.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example I A Basic Procedure for Cells Derived from Tissue Culture

HeLa cells are used as an exemplary cell type of cells that are suitablefor treatment using the compositions and methods described herein.However, the invention is in no way limited to the exemplary cell type.It is expected that the compositions and methods apply to all celltypes. One of ordinary skill would, in light of the disclosure, expectall other cells types to be amenable to the methods of the presentinvention.

To demonstrate the basic methods for cells derived from tissue culture,HeLa cells were grown in Dulbecco's Modified Eagle Medium with 10% fetalbovine serum in a tissue culture flask to 50 to 75% confluency. Themedium was removed and then the cells were incubated with trypsin (0.05%trypsin, 0.53 mM EDTA) for 10 minutes at 37° C. Trypsin was inactivatedby re-suspending the cells in medium with 10% fetal bovine serum. Cellconcentration was determined with a hemacytometer and then the volume,calculated to contain 6 million cells, was centrifuged at 3,000 rpm for5 minutes. The medium was removed and the cells were washed once with 1ml of cold phosphate buffered saline (PBS). The cells were resuspendedin 30 μl of PBS and dilutions were made in PBS using the stock solutionof 200,000; 100,000; 50,000; 25,000; 10,000; 2,000; 400; 80; and 20cells/μl. Five μl of each dilution were added to 95 μl of Cell LysisBuffer [0.2 mg/ml proteinase K (Ambion, Inc., #2546), 1 mM CaCl₂, 3 mMMgCl₂, 1 mM EDTA, 1% Triton X100, 10 mM Tris pH 7.5] such that the finalcell concentrations were 10,000; 5,000; 2,500; 1,250; 500; 100; 20; 4and 1 cells/μl. Two μl of the enterovirus Armored RNA® control (AmbionRNA Diagnostics, #42050; Pasloske, 1998) may be included in the CellLysis Buffer as a positive control to a final concentration of ˜40,000copies/O. The EV Armored RNA® Primer and probe sequences were asfollows: Forward 5′-GATTGTCACCATAAGCAGC-3′ (SEQ ID NO. 1); Reverse5′-CCCCTGAATGCGGCTAATC-3′ (SEQ ID NO. 2); TaqMan Probe:5′-(FAM)-CGGAACCGACTACTTTGGGTGTCCGT-(TAMRA)-3′ (SEQ ID NO. 3). Thesamples were incubated at 75° C. for 10 minutes and then cooled to 37°C. DNase I (Ambion, Inc., #2222) was added to the cell lysate to aconcentration of 0.02 to 0.04 U/μl, incubated at 37° C. for 15 to 30minutes and then incubated at 75° C. for 5 minutes to inactivate theDNase I. The cell lysate is now compatible for reverse transcription andPCR.

Reverse Transcription Followed by PCR

Five μl of the cell lysate from above was added to 2 μl of randomprimers, 4 μl dNTP mix (2.5 mM each) and 5 μl of RNase-free water(Ambion, Inc., #9932). The mixture was incubated at 75° C. for 3 minutesand cooled to room temperature. Two μl 10×RT buffer (500 mM Tris pH 8.3,750 mM KCl, 30 mM MgCl₂, 50 mM DTT), 1 μl (10 U/μl) of placental RNaseInhibitor (#2687, Ambion, Inc.), and 1 μl of MMLV-RT (25 U/μl) wereadded and the reaction was incubated at 42° C. from 15 minutes to 60minutes to synthesize cDNA. Negative control reactions were includedthat do not include MMLV-RT to assess the level of genomic DNAcontamination. Reactions that do not include MMLV-RT should not generateany detectable signal during PCR. The reverse transcription reaction wasincubated at 92° C. for 10 minutes to inactivate the MMLV-RT.

For PCR, 5 μl of the cDNA were combined with 1 unit of SuperTaqPolymerase (Ambion, Inc.), 2.5 μl 10×PCR buffer (100 mM Tris-Cl pH 8.3,500 mM KCl, 8% glycerol, 0.1% Tween 20), 2 μl dNTP mix (2.5 mM each), 3μl 25 mM MgCl₂, 1 μl of the primer pair (10 μM mixture of the forwardand reverse primers) and 1 μl of the TaqMan® probe (2 μM), 0.5 μl 50×ROXStandard, 8.8 μl RNase-free water (Ambion, Inc.). Human GAPDH Primer andprobe sequences were employed as follows: Forward:5′-GAAGGTGAAGGTCGGAGT-3′ (SEQ ID NO. 4); Reverse:5′-GAAGATGGTGATGGGATTTC-3′ (SEQ ID NO. 5); TaqMan Probe:5′-(FAM)-CAAGCTTCCCGTTCTCAGCC-(TAMRA)-3′ (SEQ ID NO. 6). The reactionswere placed in an ABI 7700 Prism thermocycler and ran using followingprofile: 94° C., 2 minutes; [94° C., 20 seconds; 60° C., 20 seconds]×40cycles.

GAPDH was detectable in all samples that included HeLa cells. A plot ofthe threshold cycle (Ct value) against cell concentration was linear upto 2,500 cells/μl. GAPDH signal was readily detected at cellconcentrations greater than 2,500 cells/μl but a slight inhibition ofthe reactions was observed. FIG. 1. In addition, the Ct value for theenterovirus Armored RNA® was unchanged in all of the cell concentrationsup to 2,500 cells/μl indicating that there was no inhibition of thereverse transcriptase or PCR up to 2,500 cells/(11, which was inagreement with the GAPDH data. FIG. 1.

The cDNA synthesis reactions that did not include MMLV-RT (MMLV-RT minuscontrol) did not generate any signal in the amplification reaction,demonstrating that genomic DNA was degraded to undetectable levels andthat signals produced by RT-PCR were attributable solely to theamplification of the cDNA.

Example II The Invention Functions with Multiple Cell Lines

In order to show the applicability of the invention across cell typesHeLa S3, MCF-7, COS-7, CHO-K1, and J558 cells were grown to 50-75%confluency in appropriate growth media. The cells were harvested bytrypsin, re-suspended in growth medium and counted with a hemacytometer.Two million of each cell type were collected and the cells were pelletedby centrifugation (3,000 rpm for 5 minutes). The cells were washed with1×PBS (Ambion, Inc.) and pelleted again by centrifugation (3,000 rpm for5 minutes). The cells were resuspended in 40 μl 1×PBS and four 1:5dilutions were made in PBS. Five μl of each cell suspension was added to95 μl Cell Lysis Buffer for final cell concentrations of 2,500; 500;100; 20; and 4 cells/μl in the Cell Lysis Buffer. The cells were lysed,DNase I treated, and reverse transcribed followed by PCR as in EXAMPLEI.

In each cell line, β-actin mRNA was detected by real-time PCR at eachcell concentration. ß-actin primer and probe sequences were employed asfollows: Forward: 5′-TCACCCACACTGTGCCCATCTACGA-3′ (SEQ ID NO. 7);Reverse: 5′-CAGCGGAACCGCTCATTGCCAATGG-3′ (SEQ ID NO. 8); TaqMan Probe:5′-(FAM)-ATGCCC-X(TAMRA)-CCCCCATGCCATCCTGCGTp-3′ (SEQ ID NO. 9) where Xindicates a linker-arm nucleotide, and p indicates phosphorylation. ThecDNA synthesis reactions that did not include MMLV-RT (MMLV-RT minuscontrol) did not generate any signal in the amplification reactionproving that the genomic DNA was degraded to undetectable levels andthat signals produced by RT-PCR were attributable solely to theamplification of the cDNA in each cell line.

Example III Use of Methods on Fixed Cells from Tissue Culture

To demonstrate the methods of the invention on fixed cells from tissueculture, HeLa S3 cells were grown to 75% confluency in Dulbecco'sModified Eagle Medium with 10% fetal bovine serum. The cells wereharvested as described in EXAMPLE I. Approximately 6 million cells werecollected and pelted (3,000 rpm for 5 minutes). The cells werere-suspended in 0.5 ml of 1×PBS. A volume of 0.5 ml of a solutioncontaining 2% formalin in PBS was added to make a final concentration of1% formalin. The cells were vortexed and placed at 4° C. for 1 hour.Subsequently, the cells were formalin fixed.

The cells were pelleted to remove the formalin. The cells wereresuspended in PBS and washed again to remove trace amounts of formalin.The cells were re-suspended in 120 μl PBS and four 1:5 dilutions weremade. Five μl of each cell suspension was added to 95 μl cells lysisbuffer for a final cell concentration of 2,500; 500; 100; 20 and 4cells/μl. The same procedure was performed for cells that were notformalin fixed. The cells were lysed, DNase I treated, reversetranscribed and followed by PCR as in EXAMPLE I.

GAPDH was detected in both the fixed and unfixed cells. A plot of thethreshold cycle (Ct value) against cell concentration was linear up to2,500 cells/μl for both the fixed and unfixed cells FIG. 2. The Ctvalues for both sets were nearly identical for both sets.

The cDNA synthesis reactions that did not include MMLV-RT (MMLV-RT minuscontrol) did not generate any signal in the amplification reactionproving that the genomic DNA was degraded to undetectable levels andthat signals produced by RT-PCR were attributable solely to theamplification of the cDNA.

The effect of fixing the cells up to 24 hours with a formalinconcentration as high as 4% was tested. There was about a 1 Ct valueshift between the fixed cells and the cells that were not fixed. Thiswas most likely due to loss of cells from washing the cells twice morewith 1×PBS to remove the formalin.

Those of skill would, based on this study, expect that this sameprocedure to function equally well with other types of common fixativessuch as glutaraldehyde, acetic acid/ethanol (3:1), Carnoy's fixative,Bouin's fixative, and Osmium tetroxide fixative.

Example IV Use of Methods on Fixed Cells Selected by Laser CaptureMicrodissection

To demonstrate the methods of the invention on fixed cells selected bylaser capture microdissection, frozen sections of mouse kidney embeddedwith OCT media (Tissue-Tek), were fixed and stained withHematoxylin-Eosin. Sections of 5 to 10 μm were produced with a cryostat.Areas of 0.04, 0.25, 0.6 and 1.0 mm² of tissue were captured by lasercapture microdissection using an Arcturus PixCell II™ system, each on adifferent cap. The thin layer of plastic containing the tissue sampleswas removed and placed into an 0.5 ml centrifuge tube containing 100 μlCell Lysis Buffer and 2 μl of the Armored RNA® control. The tubes wereincubated at 75° C. for 10 minutes. The lysates were then subjected toDNase I treatment, reverse transcription, and PCR as described inEXAMPLE I. Primers and probes to detect a cyclophilin sequence as wellas the Armored RNA® control were used in the PCR. Cyclophilin Primer andProbe sequences that may be employed were as follows: Forward:5′-CCATCGTGTCATCAAGGACTTCAT-3′ (SEQ ID NO. 10); Reverse: 5′-CTT GCC ATCCAG CCA GGA GGT CTT-3′ (SEQ ID NO. 11); TaqMan Probe:5′-(FAM)TGGCACAGGAGGAAAGAGCATCTATG-(TAMRA)-3′ (SEQ ID NO. 12). ACyclophilin signal was detected in all samples. The Armored RNA® controlran alongside the cyclophilin reactions indicated that there was noinhibition from the tissue samples.

Example V Multi-Well Format for Gene Expression Analysis

To demonstrate the methods of the invention in a multi-well format forgene expression analysis, cells were grown overnight in 0.2 ml DME mediawith 10% FBS with equal number of cells in each well in a 96 wellculture plate (Falcon). The media was removed and the wells were washedwith 0.2 ml 1×PBS, and the PBS was removed. One hundred μl of Cell LysisBuffer was added to each well. The plate was then moved to a heatingtile set to 75° C. and let stand for 10 minutes. To fully inactivate theproteinase K in the lysis, 2 μl of 0.1 M PMSF in DMSO was added to eachsample and incubated at room temperature for two minutes. Two μl DNase Iwas added to each sample and the plate was incubated at 37° C. for 15minutes while shaking. The plate was then moved again to the 75° C.heating tile for 5 minutes to inactivate the DNase I. One-step RT-PCRwas performed on each lysate in a 96-well PCR plate as described inEXAMPLE IX.

In each sample GAPDH was detected by real-time PCR. An analysis of theCt values for each sample gives a mean of 17.28 with a standarddeviation of 0.524. This gives a CV of 3.03% with a high of 18.88 and alow of 15.90.

If one is using a heating tile that can be set to a higher temperaturesuch, as 95° C., then it is possible to incubate the cells on theseheating tiles to inactivate the proteinase K instead of adding PMSF. Itis important that the samples themselves actually reach 75° C. whenusing proteinase K as the protease (other proteases may be inactivatedat lower temperatures). If not, then some of the protease may remainactive to digest the reverse transcriptase when the sample was incubatedfor cDNA synthesis. Such an event would lessen or completely diminish asignal from the sample.

Example VI Use of Other Reverse Transcriptases in Context of Methods

MMLV-RT is one of the most commonly used reverse transcriptases bymolecular biologists. However, there are other reverse transcriptasesthat function in the invention. For example, Avian Myelogenous Virusreverse transcriptase (AMV-RT; Retzel, 1980) and the Tth DNA polymerase,which also has reverse transcriptase activity, can each synthesize cDNA.Further, the DNA polymerase has reverse transcriptase activity if Mn⁺²is provided in the buffer (Myers, 1991) and can be used to generate cDNAfrom a cell lysate following the protocol of the invention. Those ofskill in the art will understand that the above-described nucleic acidpolymerases and any other nucleic acid polymerases having reversetranscriptase activity may be adaptable to the protocols of theinvention.

Example VII Different Concentrations of Proteinase K and Proteases Otherthan Proteinase K Function in Context of Methods of the Invention

Using the procedure listed in EXAMPLE I, proteinase K has been used atconcentrations of 25 and 500 μg/ml. The results have been essentiallythe same as using the 200 μg/ml concentration that was used in thestandard protocol.

Proteases are classified into several groups based on the mechanism ofcatalysis. Serine proteases include but are not limited to Trypsin,Chymotrypsin, Elastase, Subtilisin, Streptogrisin, Thermitase,Aqualysin, and carboxypeptidase A, D, C, or Y. Cysteine proteasesinclude but are not limited to Papain and Clostripain. Acid Proteasesinclude but are not limited to Pepsin, Chymosin, and Cathepsin.Metalloproteases include but are not limited to Pronase, Thermolysin,Collagenase, Dispase, various aminopeptidases, and Carboxypeptidase A,B, E/H, M, T, or U. In some embodiments of the invention, theseproteases or combination thereof could be used in place of or inaddition to proteinase K. It is possible that a mixture of proteasescould be used instead of a single protease to generate a cell lysatecompatible with reverse transcription and PCR. In certain embodiments aprotease or protease mixture may be used simultaneously with a nuclease,e.g., a DNase such as DNas I, or any other catabolic enzyme.

Example VIII Tissue Samples

Another type of sample that may be used in regard to the invention is apiece of tissue consisting of cells ranging from hundreds to thousands.One such tissue or organ may be leech ganglia. Another sample type maybe a patient needle biopsy that often consists of thousands of cells. Abiopsy could be processed by the inventive methods and PCR amplified tomake a diagnosis or prognosis by measuring the expression of certaingenes. Another sample may be leukocytes or lymphocytes isolated from ablood sample. Plasma fractionated from a blood sample may be used inthis invention to detect a virus such as HIV or HCV. Another sample maybe whole blood itself.

Example IX Coupling of Invention with One-Step RT-PCR

Methods of the invention can be used in a one-step RT-PCR reaction wherethe MMLV-RT and Taq polymerase were combined in a one tube, one buffersystem. For example, HeLa S3 cells were grown, harvested, lysed, andDNase treated as in EXAMPLE I. Cell lysate concentrations of 1, 4, 20,100, 500, 1,250, 2,500, 5,000, and 10,000 cells/μl were made and DNasetreated as in EXAMPLE I. Five μl of each lysate was added to 2.5 μl10×RT buffer (500 mM Tris pH 8.3, 750 mM KCl, 30 mM MgCl₂, 50 mM DTT), 1μl (10 U/μl) of placental RNase Inhibitor (cat. #2687, Ambion, Inc.), 1μl of MMLV-RT (25 U/μl), 4 μl dNTP mix (2.5 mM each), 0.5 μl 50×ROXstandard (Ambion, Inc.), 1 μl PCR primer mix (10 μM mix of forward andreverse primers), 1 μl TaqMan probe (2 μM), 0.2 μl SuperTaq (Ambion,Inc.), and 8.8 μl RNase-free water (Ambion, Inc.). The reactions wereplaced in an ABI 7700 Prism thermocycler and the following profile wasran: 42° C., 15 minutes; 94° C., 2 minutes; [94° C., 20 seconds; 60° C.,20 seconds]×40 cycles.

GAPDH was detected in all samples that included HeLa cells. A plot ofthe threshold cycle (Ct value) against cell concentration was linear upto 2,500 cells/μl. GAPDH signal was detected at higher cellconcentrations but inhibition of the reactions by higher cellconcentrations was indicated by Ct values increasing at the higher cellconcentrations compared to the lower concentrations.

The cDNA synthesis reactions that did not include MMLV-RT (MMLV-RT minuscontrol) did not generate any signal in the amplification reactionproving that the genomic DNA was degraded to undetectable levels andthat signals produced by RT-PCR are attributable solely to theamplification of the cDNA.

Example X Use of Invention without the DNase Treatment

The DNase I step in some aspects of the invention is not necessary ifthe PCR primers used will not amplify genomic sequences. This can bedone by designing primers that span an intron within the gene ofinterest. This greatly reduces the total time it takes to complete theprocedure of the invention because the DNase I treatment can beeliminated.

For example, HeLa S3 cells were grown, harvested and lysed as describedin EXAMPLE I. Reverse transcription followed by PCR was performed, againas in EXAMPLE I. A one-step RT-PCR procedure on the same lysates wasperformed as described in EXAMPLE IX using primers and probe for DDPK.DDPK Primer and Probe sequences, can be, for example: Forward:5′-CTGGCCGGTCATCAACTGA-3′ (SEQ ID NO. 13); Reverse:5′-ACAAGCAAACCGAAATCTCTGG-3′ (SEQ ID NO. 14); TaqMan Probe:5′(FAM)-AATGCGT-(TAMRA)-CCTGAGCAGCAGCCCp-3′ (SEQ ID NO. 15). DDPK wasdetected by real time PCR, and the reverse transcriptase minus reactionswere negative.

Example XI RNA Amplification

There are many cases where researchers have a limited amount of sampleand the RNA isolated from the sample is not enough to perform theirdesired assay. The technique that this applies to most often isproducing a labeled nucleic acid from the isolated RNA and thenhybridizing the labeled nucleic acid to a microarray. The signalsproduced at each of the addresses of the microarray indicate the levelof expression for each of the genes on the array. Thus, a snapshot istaken of the abundance for each of the genes probed by the array.

Typically, the starting material for amplifying RNA is a minimum of ˜10ng of total RNA from the sample. Next, the RNA is reverse transcribed inthe presence of an oligonucleotide primer that encodes an RNA polymerasepromoter such as a T7 phage promoter. In the procedure by Kacian (U.S.Pat. No. 5,554,516), the material is now transcribed by T7 RNApolymerase to synthesize RNA. In the procedure by Phillips (1996), asecond strand of cDNA is produced and then the double-stranded DNA istranscribed by a phage polymerase. Ambion, Inc. produces the MessageAmpkit (Cat. #1750) based on the procedure of Phillips (1996). Purifiedtotal RNA or poly(A)RNA is the recommended substrate for the MessageAmpkit.

Cell lysates generated by the Cells-to-cDNA II™ procedure weredemonstrated to be suitable substrate for the MessageAmp kit. Forexample, K562 cells at concentrations of 200, 600 and 2,000 cells/μlwere incubated at 75° C. for 10 minutes in a Cell Lysis Buffer comprisedof 50 mM Tris pH 8.3, 75 mM KCl, 5 mM DTT, 1 mM EDTA, 1% TX-100, and 200μg/ml proteinase K. No DNase I treatment was performed because it is notneeded in this application. Five μl of each cell lysate concentrationwas used as the template in the MessageAmp procedure. RNA was amplifiedfrom 860 to 3,000 fold with this procedure (TABLE 1).

TABLE 1 Fold-Amplification of RNA from a Cell Lysate Generated by theCells-to- cDNA II ™ method using the MessageAmp kit. Quantity of RNANumber of Cells *Quantity After Added to of RNA in MessageAmp MessageAmpthe Reaction Amplification Fold-Amplification Reaction as Lysate (ng)(μg) of Cellular RNA 1,000 5 15 3,000 3,000 15 25 1,667 10,000 50 43 860*Assume 5 pg of total RNA per cell

Example XII Automation and Monitoring the Effects of Drug Treatment

Cells were grown overnight in 0.2 ml DME media with 10% FBS with equalnumber of cells in each well of a 96-well plate. After an overnightincubation, phorbol myristate acetate (PMA) is added to finalconcentrations of 100, 10, 1, 0.1, and 0 nM in the growth medium inreplicates of eight. The cells were incubated at 37° C. for 24 hours.The 96-well plate and lid were placed on the Packard MultiPROBE® II HTLiquid Handling System from PerkinElmer Life Sciences on the proper deckpositions. All proceeding steps were entered into the WinPrep forautomation. The growth medium was removed and the cells washed with1×PBS. A volume of 0.1 ml Cell Lysis Buffer is added to each well. Theplate was moved to a heating tile, for example, a tile at 75°, andincubated for 10 minutes. The plate was then moved to a shaker platformand 2 μl 100 mM PMSF in DMSO was added to each well to inactivate theproteinase K. PMSF is a serine protease inhibitor that does not inhibitreverse transcriptase or Taq polymerase. The plate was shaken for 2minutes. PMSF can be used when the heating tile cannot heat the sampleto 75° C. to inactivate the proteinase K. Two μl DNase I (2 U/μl) wasadded to each well and the plate was moved to a 37° C. heating tile. Theplate was incubated for 15 minutes while shaking. The plate was thenmoved to a heating tile, for example, a tile at 75° C., and incubatedfor 5 minutes to inactivate the DNase I. The protocol for one stepRT-PCR found in EXAMPLE IX was followed so that 20 μl aliquots of themaster mix were added to a 96-well PCR plate. Five μl of lysate wasadded to each well. The plate was ready for one-step RT-PCR.

The one-step RT-PCR was run with primers and TaqMan probes for bothtissue plasminogen activator (t-PA) and 18S ribosomal RNA with astandard curve ran for each set. tPA Primer and Probe sequences were:Forward: 5′-GGCGCAGTGCTTCTCTACAG-3′ (SEQ ID NO. 16); Reverse:5′-TAGGGTCTCGTCCCGCTTC-3′ (SEQ ID NO. 17); TaqMan Probe:5′-(FAM)-TTCTCCAGACCCACCACACCGC-(TAMRA)-3′ (SEQ ID NO. 18); 18S Primerand Probe sequences: Forward: 5′TCAAGAACGAAAGTCGGAGG3′ (SEQ ID NO. 19);Reverse: 5′GGACATCTAAGGGCATCACA3′ (SEQ ID NO. 20); TaqMan Probe:5′-(FAM)-TGGCTGAACGCCACTTGTCCCTCTAA-(TAMRA)-3′ (SEQ ID NO. 21). Thereal-time PCR data shows there was about a 29-fold stimulation of t-PAwith concentrations of 100 and 10 nM when the values were normalized tothe levels of 18S rRNA which is assumed to be constant during differentexperimental conditions. FIG. 3.

In a similar type of experiment, cells were treated with shortinterfering (si)RNA to down regulate specific genes (Elbashir, 2002).Three thousand cells grown in a 96-well dish were transfected withchemically synthesized, 125 nM gene-specific siRNAs against CDC-2,c-jun, survivin, and GAPDH or a negative control (NC1) 24 hours afterplating using Oligofectamine transfection reagent (Invitrogen). After 48hours, cells were processed according to the Cells-to-cDNA II™ automatedprotocol, and analyzed by real-time one-step RT-PCR on an ABI 7900 forthe indicated genes and normalized to 18S rRNA. Gene expression wascalculated as a percentage of gene expression compared with the negativecontrol siRNA. Experiments were performed in replicates of eight. Usingthe cell lysate produced by the Cells-to-cDNA II™ procedure and one-tubeRT-PCR, gene expression for each of the target genes was determined tobe reduced by more than 70% (FIG. 4).

Example XIII Multiple Enzymes Used to Produce the Cell Lysate

In addition to proteases, other types of enzymes may be included in theCell Lysis Buffer, such as enzymes to digest nucleic acids, sugars,fats, connective tissue (collagen and elastin) and DNA. These enzymesmay be more important with regard to EXAMPLE VIII where the system maybe used for small quantities of tissue. A combination of enzymes mayenhance the digestion process and also destroy the macromolecules thatinhibit enzymatic reactions.

For example, the initial lysis step containing proteinase K was combinedwith the DNase treatment and the cellular DNA was digested tosubstantially reduce the signal generated in the reverse transcriptaseminus reactions. This result was unexpected since it was thought thatthe proteinase K would digest the DNase I before the DNase could fullydigest the cellular DNA. By combining these two enzymatic reactions, theCells-to-cDNA II™ process is further streamlined and only a singleincubation event is required to produce a cell lysate compatible withreverse transcription or one-step RT-PCR.

The Cell Lysis Buffer was made with proteinase K concentrations of 20 to200 μg/ml and placed on ice. DNase I was added to the Cell Lysis Buffersat 0.1 U/μl and then the Cell Lysis Buffers were kept on ice while thecells were harvested. Cells were added to the different Cell LysisBuffers at concentrations of 4 to 2,500 cells/μl. The samples wereheated in a thermocycler at 60° C. for 10 minutes to give the DNase timeto digest the cellular DNA before inactivating the proteinase K andDNase enzymes at 75° C. for 10 minutes. The same cells were also treatedusing the sequential method of EXAMPLE I. One-step real-time RT-PCR forGAPDH (as in EXAMPLE IX) was performed on these lysates. The differencebetween the RT minus and RT plus reactions were 18 Ct (˜20,000×)indicating that the DNase I was active in the combined format and thatthe proteinase K did not digest the DNase until it had degraded nearlyall of the cellular DNA (TABLE 2).

TABLE 2 Comparison of the signal obtained for GAPDH by real-time RT-PCRand PCR (RT−) using a combined or sequential proteinase K and DNase Itreatments of HeLa S3 cells. Cell Concentration Combined (Ct) Sequential(Ct) (cells/μl) RT(+) RT(−) RT(+) RT(−) 4 21.3 40 21.4 40 20 17.6 4018.1 40 100 16.9 31.9 15.7 33.5 500 14.6 33.3 14.1 36.7 2,500 12.9 31.512.7 31.7

Example XIV Addition of Cell Lysis Buffer Directly to Tissue CultureMedium

Typically, the Cells-to-cDNA II™ procedure involves washing the cellsgrown in tissue culture with phosphate buffered saline (PBS), primarilyto remove the serum from the sample that was in the growth medium. Theserum can inhibit downstream enzymatic reactions like reversetranscription and PCR.

If the cells can be grown in serum-free medium, then the cell-washingstep may be bypassed entirely. As such, the processing time will bedecreased. Also, this procedure makes the handling of cells grown insuspension much easier because centrifugation is no longer required forwashing. Drosophila cells are commonly grown in serum-free medium andtherefore, the efficiency of the Cells-to-cDNA II™ procedure on thesecells, if the cell washing step were omitted, was tested.

Schneider L2 Drosophila cells were grown to confluency in Drosophila-SFM(serum free medium). The cells were harvested in SFM and diluted toconcentrations of 6,500; 1,300; 260; 52; and 10.4 cells/μl in SFM.Eighty μl of each cell concentration was added to a 0.5 ml tube to whichtwo volumes (160 μl) of Cell Lysis Buffer was added. These samples werethen taken through the Cells-to-cDNA II™ protocol including the DNase Idigestion. The cell lysates were then used in one-step, real-time RT-PCRto detect Ubiquitin (accession #M22428) and analyzed using the ABIPRISM® 7900 HT Sequence Detection System Forward Primer:5′-CACGCATCTTGTTTTCCCAAT-3′ (SEQ ID NO:22); Reverse Primer:5′-CTCGAGTGCGTTCGTGATTTC-3′ (SEQ ID NO:23); TaqMan Probe:5′-AATTGGCATCAAAACGCAAACAAATC-3′ (SEQ ID NO:24). Lysate volumes of 5 μlwere used in each of the 20 μl reactions.

It was found that all cell concentrations generated a PCR signal andthat they were in the linear range. In addition, no signal was detectedin the RT minus reactions indicating that the DNase treatment waseffective in the presence of the SFM medium.

Example XV Use of an Exemplary Kit for Producing cDNA from MammalianCells in Culture

Components of an exemplary kit for the preparation of cDNA frommammalian cells in culture may include one or more of the followingreagents: 1×PBS (pH 7.4); Cell Lysis II Buffer (10 mM Tris pH 7.5, 3 mMMgCl₂, 1 mM CaCl₂, 1 mM EDTA pH 8.0, 1% Tx-100 and 200 ug/ml ProteinaseK); DNase 1 (2 U/μl); 10×RT Buffer (500 mM Tris pH 8.3, 750 mM KCl, 30mM MgCl2, 50 mM DTT); M-MLV Reverse Transcriptase; RNase Inhibitor (10U/μl); dNTP Mix (2.5 mM each dNTP); Random Decamers (50 μM); Oligo(dT)₁₈Primers (50 μM); Nuclease-free water; RNA control, e.g., Armored RNAcontrol; control RNA primer pair, e.g., Armored RNA primer pair (10 μMeach); and an endogenous Primer Pair (5 μM each). The kit components aresupplied in suitable containers under suitable conditions for shippingor storage. The parameters for use of the kit components are describedherein and may be used to produce cDNA from mammalian cells in culturewithout the isolation of mRNA. In certain embodiments, the cDNA producedmay be used in a variety of assays or procedures including, but notlimited to PCR amplification or automated PCR amplification and analysisin multiwell formats and RNA amplification.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Brady G, Iscove N N. Construction of cDNA libraries from single    cells. Methods Enzymol. 225: 611-623, 1993.-   Busche S, Gallinat S, Bohle R-M, Reinecke A, Seebeck J, Franke F,    Fink L, Zhu M, Sumners C, Unger T. Expression of angiotensin AT1 and    AT2 receptors in adult rat cardiomyocytes after myocardial    infarction: a single-cell reverse transcriptase-polymerase chain    reaction study. J. Am. Pathol. 157: 605-611, 2000.-   Elbashir S M, Harborth J, Weber K, Tuschl T. Analysis of gene    function in somatic mammalian cells using small interferingRNAs.    Methods 26: 199-213, 2002.-   Fink L, Kinfe T, Seeger W, Ermert L, Kummer W, Bohle R M.    Immunostaining for cell picking and real-time mRNA quantitation.    Am. J. Pathol. 157: 1459-1466, 2000a.-   Fink L, Kinfe T, Stein M M, Ermet L, Hanze J, Kummer W, Seeger W,    Bohle R M. Immunostaining and laser-assisted cell picking for mRNA    analysis. Laboratory Invest. 80; 327-333, 2000b.-   Klebe R J, Grant G M, Grant A M, Garcia M A, Giambernardi T A,    Taylor G P. R T-PCR without RNA isolation. BioTechniques 21:    1094-1100, 1996.-   Lockhart D J, Dong H, Byrne M C, et al. Expression monitoring by    hybridization to high-density oligonucleotide arrays. Nat.    Biotechnol. 14: 1675-1680, 1996.-   Mesink E, van de Locht A, Schattenberg A, Linders E, Schaap N,    Geurts van Kessel A, de Witte T. Quantitation of minimal residual    disease in Philadelphia chromosome positive chronic myeloid leukemia    patients using real-time quantitative RT-PCR. Br. J. Haematol. 102:    768-774, 1998.-   Myers T W, Gelfand D H. Reverse transcription and DNA amplification    by a Thermus thermophilus DNA polymerase. Biochemistry 30:    7661-7666, 1991.-   O'Leary T J. Reducing the impact of endogenous ribonucleases on    reverse transcription-PCR assay systems. Clin. Chem. 45: 449-450,    1999.-   Pasloske B L, WalkerPeach C R, Obermoeller R D, Winkler M, DuBois    D B. Armored RNA technology for the production of ribonuclease    resistant viral RNA controls and standards. J. Clin. Microbiol. 36:    3590-3594, 1998.-   Phillips J, Eberwine J H. Antisense RNA amplification: a linear    amplification method for analyzing the mRNA population from single    living cells. Methods 10: 283-288, 1996.-   Retzel E F, Collett M S, Faras A J. Enzymatic synthesis of    deoxyribonucleic acid by the avian retrovirus reverse transcriptase    in vitro: optimum conditions required for transcription of large    ribonucleic acid templates. Biochemistry 19: 513-518, 1980.-   Sumida A, Yamamoto I, Zhou Q, Morisaki T, Azuma J. Evaluation of    induction of CYP3A mRNA using the HepG2 cell line and reverse    transcription-PCR. Biol. Pharm. Bull. 22: 61-65, 1999.-   Su S, Vivier R G, Dickson M C, Thomas N, Kendrick M K, Williamson N    M, Anson J G, Houston J G, Craig F F. High-throughput R T-PCT    analysis of multiple transcripts using a microplate RNA isolation    procedure. BioTechniques 22: 1107-1113, 1997.-   Tang J, Hu M, Lee S, Roblin R. A polymerase chain reaction based    method for detecting Mycoplasma/Acholeplasma contaminants in cell    culture. J. Microbiol. Methods 39: 121-126, 2000.-   Yan L, Kaczorowski G, Kohler M. One-tube protocol for single-cell    reverse transcriptase-polymerase chain reaction. Anal. Biochem. 304:    267-270, 2002.-   Roblin III R O, Hu M, Tang J S, Sunmin L. Mycoplasma polymerase    chain reaction testing system using a set of mixed and single    sequence primers. U.S. Pat. No. 5,693,467.-   Van Gelder R N, von Zastrow M E, Barchas J D, Eberwine J H.    Processes for genetic manipulations using promoters. U.S. Pat. No.    5,891,636.-   Kacian D L, McAllister D L, McDonough S H, Dattagupta N. Nucleic    acid sequence amplification method, composition and kit. U.S. Pat.    No. 5,554,516.-   Reynolds M A, Ruvolo M, Arnold Jr. L J. Combined polynucleotide    sequence as discrete assay endpoints. U.S. Pat. No. 6,316,608.

1.-70. (canceled)
 71. A method for producing cDNA from at least onebiological unit containing RNA, wherein the biological unit is a cellobtained from cell culture, from a tissue sample or from a body fluid,comprising: preparing an admixture comprising the biological unitproteinase K and a deoxyribonuclease; incubating the admixture underconditions where the proteinase K and deoxyribonuclease are active;raising the temperature of the admixture containing the activeproteinase K and deoxyribonuclease to inactivate the proteinase K anddeoxyribonuclease; incubating the admixture with at least one reversetranscriptase under conditions to allow reverse transcription to producecDNA; and further comprising amplifying the cDNA by real-time PCR. 72.The method of claim 71 wherein the deoxyribonuclease comprises DNase 1.73. The method of claim 71, wherein the biological unit is a cellobtained from cell culture.
 74. The method of claim 71, wherein thebiological unit is a cell obtained from a tissue sample.
 75. The methodof claim 71, wherein the biological unit is a eukaryotic cell.
 76. Themethod of claim 75, wherein the eukaryotic cell is a human cell.
 77. Themethod of claim 71 wherein the biological unit is a prokaryotic cell.78. The method of claim 71, wherein the biological unit is a fungalcell.
 79. A method for producing cDNA for quantification of RNA from atleast one biological unit containing RNA, wherein the biological unit isa cell obtained from cell culture, from a tissue sample or from a bodyfluid, comprising: preparing an admixture comprising the biologicalunit, a deoxyribonuclease and at least one catabolic enzyme, wherebysaid enzyme is at least proteinase K; incubating the admixture underconditions where the proteinase K and the deoxyribonuclease are active;raising the temperature of the admixture to inactivate the proteinase Kand deoxyribonuclease; incubating the admixture with at least onereverse transcriptase under conditions to allow reverse transcription toproduce cDNA.
 80. The method of claim 79, wherein the biological unit isa cell obtained from cell culture.
 81. The method of claim 80, furthercomprising amplifying the cDNA by real-time PCR.
 82. The method of claim79, wherein the biological unit is a cell obtained from a tissue sample.83. The method of claim 79, wherein the biological unit is a eukaryoticcell.
 84. The method of claim 83, wherein the eukaryotic cell is a humancell.
 85. The method of claim 79, wherein the biological unit is aprokaryotic cell.
 86. The method of claim 79, wherein the biologicalunit is a fungal cell.